Participants
Twenty-four healthy volunteers (12 females) aged 21–30 years old, (mean ± standard deviation: SD, 22 ± 2 years) participated in the experiment. None of the participants had any history of neurological and/or musculoskeletal disorders. The number of participants was decided based on a previous study that investigated the modulation of CMC by NMES [18]. All participants were right-foot dominant as confirmed by the foot-dominant test [20]. All participants provided written informed prior to participation. The experimental protocol was approved by the Ethical Review Board of Yamagata Prefectural University of Health Science in Japan and conformed to the ethical standards laid down in the 1964 Declaration of Helsinki.
NMES
NMES was delivered with a stimulator (SEN-3401, Nihon Kohden, Tokyo, Japan). The first stimulation frequency for NMES was defined as beta-band frequency NMES (β-NMES) and was estimated from the peak of the beta-band CMC frequency (mean ± SD, 21 ± 5 Hz), while participants performed isometric dorsiflexion tasks before the stimulation. The second stimulation frequency for NMES was defined as the fixed frequency of NMES (f-NMES) and consisted of a train of 10 pulses at 100 Hz generated every 2 s [21]. The stimulation was delivered to the right common peroneal nerve (CPN) for 20 min [22]. The stimulation intensity was 95% of the motor threshold with a pulse width of 1 ms. The motor threshold was defined such that the minimum stimulation intensity evoked muscle twitch contraction of the tibialis anterior (TA) muscle.
Electroencephalographic (EEG) and electromyographic (EMG) recordings
The participants sat comfortably on a chair with their feet firmly strapped to a foot plate. EEG and EMG were recorded with Ag/AgCl electrodes. Before the attachment of electrodes, the skin was rubbed with an alcohol pad and the skin impedance was kept below 5 kΩ. EEG electrodes were placed at Cz representing the ankle muscles and at 5 cm frontal to Cz, according to the international 10–20 system of electrode placement [7]. EMG electrodes were placed over the TA muscle belly in the right lower limb with an inter-electrode distance of 2 cm. EEG and EMG data were simultaneously acquired (Neuropack MEB-2200, Nihon Kohden, Tokyo, Japan), bandpass filtered in the frequency range of 0.5–200 Hz in the case of EEG and in the range of 5–500 Hz in the case of EMG. Force signals were recorded with a force transducer (Takei Scientific Instruments Co. Niigata, Japan). EEG, EMG, and force data were recorded while the participants performed isometric dorsiflexion to maintain their exerted force as close as possible to the line that corresponded to 10% of their maximum voluntary contraction (MVC) force for 2 min [23]. All signals were converted to digital signals at a sample frequency of 5 kHz by analog-to-digital converter with a 16-bit resolution (NI USB-6363, National Instruments, Austin, TX, USA) controlled by the data-logger software LabVIEW2018 (National Instruments Co., TX, USA).
Corticomuscular coherence
CMC was estimated by EEG and EMG data following Eq. (1) [24]. CMC describes the linear association between EEG and EMG signals at each frequency of interest. It is a measure of phase consistency between signals. CMC estimates are defined over the range 0–1, where the value of zero indicates no association, and one indicates a perfect association. To calculate CMC, auto-spectra and cross-spectra were constructed by dividing signals into nonoverlapping segments. Discrete Fourier transforms were then performed on each segment and averaged. CMC was then determined as the squared modulus of the cross-spectrum for the two signals fxy(j), normalized by the product of the two auto-spectra, fxx(j), and fyy(j).
(1)
The statistical significance of CMC estimates was assessed according to an upper 95% confidence limit.
Force steadiness of ankle dorsiflexion
The right foot was strapped to the plate with the ankle in the neutral position. Participants were asked to maintain an isometric dorsiflexion at 10% MVC for 2 min. The target force line was presented in front of participants with a display. Participants were instructed to follow the target line as closely as possible with the moving red line (real-time dorsiflexion force). It was reported that the beta-band CMC was negatively correlated with the coefficient of variation [(standard deviation/mean) × 100%] of force (Force CV). If NMES changes the beta-band CMC, the changes in the beta-band CMC could be related with the changes in the exerted steady-state of ankle dorsiflexion. To investigate the exerted steadiness of dorsiflexion force during the ankle dorsiflexion task, Force CV was calculated as a measure of the control of the exerted force.
Experimental procedure
This study employed a randomized crossover design. Participants received β-NMES and f-NMES on two different days (Fig. 1). To assess beta-band CMC changes, EEG and EMG were measured before stimulation (pre) and after stimulation at 0 min (post0), 10 min (post10) and 20 min (post20). To prevent carry-over effects from the previous NMES condition, washout intervals of 3 days (or more) were included between sessions.
Complementary experiment
Beta-band CMC reflects the functional coupling between primary motor cortex and spinal motor neurons that related to corticospinal excitability [9, 25]. Corticospinal excitability was modulated by NMES [22, 26–28]. Therefore, the changes in the beta-band CMC induced by β-NMES may be related to the changes in corticospinal excitability. To address this question, 14 healthy volunteers (5 females) aged 20–32 years old, (mean ± SD, 24 ± 3 years) received β-NMES (mean ± SD, 25 ± 5 Hz) for 20 min. To assess corticospinal excitability, single-pulse TMS was delivered to the primary motor cortex responsible for motor representation of the leg with a figure-8 coil (D702) that was connected to the Magstim200 (Magstim Company, Whitland, United Kingdom). The optimal coil positioning on the hot spot of the primary motor cortex was identified for the induction of the largest motor evoked potentials (MEPs) amplitudes in the right TA. The stimulation intensity was adjusted to 120% of the active motor threshold (aMT). The aMT was defined as the minimum stimulus intensity that produced 200 µV MEPs with a probability of 50% during isometric contraction at 100 µV of TA EMG. Fifteen MEPs were recorded while the participants performed an isometric contraction with 100 µV of TA. Before the main assessment, baseline MEPs and CMC were measured to normalize the data. Following a 2-min rest period, MEPs and CMC were assessed pre, post at 0 min, 10 min (post10), and 20 min (post20). To minimize the influences of voluntary contraction during the CMC assessment, the MEPs were assessed experimentally before the CMC assessment.
Statistical analysis
Normality was assessed with the Shapiro–Wilk test. The Wilcoxon signed-rank test was used to compare pre data between the raw value of the beta-band CMC and Force CV. Friedman test was used to investigate the main effect of time (pre, post, post10, and post20) on the raw values of the beta-band CMC and Force CV. Post-hoc tests were performed using the Wilcoxon signed-rank test with Bonferroni adjustments when a significant main effect was found. Spearman’s rank correlation was used to define the relationship between the normalized beta-band CMC and the Force CV induced by stimulations in assessment time points (post, post10, and post20). The normalized beta-band CMC and normalized Force CV were calculated by the obtained data pre.
In the complementary experiment, the normalized beta-band CMC and normalized MEPs were calculated with the baseline data. Friedman’s test was used to investigate the main effect of time (pre, post, post10, and post20) on the normalized MEPs. Post-hoc tests were performed with the Wilcoxon signed-rank test with Bonferroni adjustments when a significant main effect was found. Pearson’s product moment correlation was used to investigate the relationship between the normalized MEPs and normalized beta-band CMC at assessment time points after β-NMES (pre, post, post10, and post20). P-values < 0.05 indicated statistical significance in all analyses. Statistical analyses were performed with SPSS (version 24.0, IBM Corporation, New York, NY, USA) for Windows.